: Why don't you do direct sequencing of your PCR products using the
: Sequenase 2.0 version. It has no exonuclease activity and highly processive.
: This will take the scare out of 5'-3' exonuclease activity.
Thanks for the suggestion. I didn't think it would work. I have tried
sequencing linear dsDNA with Sequenase and it didn't work. Worked great
with circular (plasmid) DNA. Apparently, it is difficult to keep linear
DNA in a denatured state during the sequencing reaction. That's why I
purify ssDNA for sequencing. Cycle sequencing also works but not as nicely
as with ssDNA as template.
If you've had good luck sequencing with Sequenase using LINEAR dsDNA as
template, I would appreciate some pointers. Thanks again.
Shiao Wang
University of Southern Mississippi